Current chemical hazard assessment for developmental toxicity is built upon globally harmonized OECD (Organization for Economic Co-operation and Development) animal test guidelines, providing a structure for chemical risk assessment and in addition encouraging the development of alternative testing strategies. Under REACH (Regulation, Evaluation and Authorization of Chemicals) guidelines, ˜60% of all animals will be used for reproductive and developmental toxicity studies (van der Jagt et al., 2004).
In order to reduce the number of experimental animals needed for developmental toxicity testing, cell based alternative test methods are being developed, such as, embryonic stem cell tests studying multiple differentiation lineages (Genschow et al., 2004; Piersma et al., 2004; Theunissen et al., 2010; zur Nieden et al., 2010), the whole embryo culture (WEC) (Piersma et al., 2004) and the zebrafish embryo test (Hermsen et al., 2011; Nagel, 2002).
An important target for developmental toxicity is the developing nervous system. Classical developmental toxicants, such as, methylmercury (MeHg), valproic acid, cyproconazole and ethanol particularly target neural development resulting in neural tube defects, craniofacial malformations, mental retardation and fetal alcohol syndrome (Alsdorf and Wyszynski, 2005; Faustman et al., 2002; Welch-Carre, 2005).
Current in vitro test systems for determining neurodevelopmental toxicity depend on assessing cellular endpoints of toxicity, such as, cell morphology (neural outgrowth, cytotoxicity, migration) (Mundy et al., 2010; Radio and Mundy, 2008) or functional parameters determined by electric conductivity (Shafer et al., 2002).
Regulatory authorities often require that compounds are tested in animals for neurodevelopmental toxicity. Such systems suffer from the disadvantage that they are laborious and costly and require large numbers of animals. In addition, the methods involve behavioral testing which is inherently variable and requires substantial efforts and experimental skills, presupposing high investments in training and equipment.
Prediction of neurodevelopmental toxicity by in vitro methods is hampered by the various mechanisms through which the toxicity can occur. We previously showed that the differentiation track algorithm can be applied to predict toxicity in the neural stem cell test (Theunissen et al., Toxicol Sci. 2011 August; 122(2):437-47). This algorithm relates gene expression changes due to compound exposure at a given time point to gene expression changes to an earlier and later time point by “normal” (unexposed) differentiation. However, until now this relied on using over a thousand genes, and no small and optimized set was available.
It is an object of the present invention to overcome or ameliorate at least some of the disadvantages of the prior art methods.